Non-small-cell lung cancer (NSCLC) accounts for approximately 80% of all lung cancers, with 1.2 million new cases worldwide each year. NSCLC resulted in more than one million deaths worldwide in 2001 and is the leading cause of cancer-related mortality in both men and women (31% and 25%, respectively). The prognosis of advanced NSCLC is dismal. A recent Eastern Cooperative Oncology Group trial of 1.155 patients showed no differences among the chemotherapies used: cisplatin/paclitaxel, cisplatin/gemcitabine, cisplatin/docetaxel and carboplatin/paclitaxel. Overall median time to progression was 3.6 months, and median survival was 7.9 months, a 1-year survival rate of 33% and a 2-year survival rate of 11 percent. A more recent randomized study of 1218 patients reported a median survival of 11 months in stage IIIB-IV patients. However, no clinical parameters can completely account for the striking differences in survival among patients with advanced disease, with some surviving years and others only a few months.
The overall five-year survival of patients with NSCLC has remained at less than 15% for the past 20 years. Stage grouping of TNM subsets (T=primary tumor; N=regional lymph nodes; M=distant metastases) permits the identification of patient groups with similar prognosis and treatment options. Five-year survival is around 25% for pathologic stage IIB (T1-2N1M0, T3N0M0), 13% for stage IIIA (T3N1M0, T1-2-3N2M0), and a low 7% for stage IIIB (T4N0-1-2M0).
Currently, cisplatin (DDP) and carboplatin are among the most widely used cytotoxic anticancer drugs. However, resistance to these drugs through de novo or induced mechanisms undermines their curative potential. These drugs disrupt DNA structure through formation of intrastrand adducts. Resistance to platinum agents such as DDP has been attributed to enhanced tolerance to platinum adducts, decreased drug accumulation, or enhanced DNA repair.
14-3-3σ is a member of the 14-3-3 superfamily that is responsible for G2 cell cycle checkpoint control in response to DNA damage in human cells. Its function has been analyzed in the human colorectal cancer cell line HCT116 (expressing 14-3-3σ and wild-type p53). Following ionizing irradiation, 14-3-3σ sequestered Cdc2/cyclin B1 complexes in the cytoplasm, thus arresting cells in G2 and preventing them from initiating mitosis before repair to their damaged DNA. Colon carcinoma cells lacking 14-3-3σ treated with adriamycin can still initiate—but do not maintain—G2 arrest, leading to mitotic catastrophe and cell death. The expression of 14-3-3σ is reduced by p53 gene inactivation and by silencing of 14-3-3σ gene via methylation of CpG islands.
By proteomic analysis, 14-3-3σ was undetectable in breast cancer samples, and hypermethylation of normally unmethylated CpG islands in the promoter region of 14-3-3σ was involved in gene silencing at the transcriptional level in breast cancers (Ferguson A T, Evron E, Umbricht C B, et al.: High frequency of hypermethylation at the 14-3-3 sigma locus leads to gene silencing in breast cancer. Proc Natl Acad Sci USA 2000; 97:6049-54). Similar effects of 14-3-3σ hypermethylation have been reported in many tumors, including lung, gastric, ovarian, prostate, and hepatocellular carcinomas.
It is known that double-stranded DNA fragments frequently occur in considerable quantities in the serum of cancer patients, with significantly higher levels found in the serum of patients with metastases. In head and neck, small-cell lung and non-small-cell lung cancers, the same microsatellite alterations detected in the tumor were also found in plasma or serum DNA (Sanchez-Cespedes M, Monzo M, Rosell R, et al. Detection of chromosome 3p alterations in serum DNA of non-small-cell lung cancer patients. Ann Oncol 1998; 9:113-6; Sozzi G, Musso K, Ratcliffe C, Goldstraw P, Pierotti M A, Pastorino U. Detection of microsatellite alterations in plasma DNA of non-small cell lung cancer patients: a prospect for early diagnosis. Clin Cancer Res 1999; 5:2689-92). Furthermore, the detection of hypermethylation in the promoter regions of tumor suppressor genes was first reported in the serum of non-small-cell lung cancer patients. Hypermethylation can be analyzed by the sensitive methylation-specific polymerase chain reaction assay, which can identify one methylated allele in 1000 unmethylated alleles (Herman J G, Graff J R, Myohanen S, Nelkin B D, Baylin S B. Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands. Proc Natl Acad Sci USA 1996; 93:9821-6).
14-3-3σ was found to be methylated in 43 percent of 60 gastric cancers and the 14-3-3σ methylation-positive human gastric cell lines MKN74 (with wild-type p53) and MKN28 (with mutated p53) were both highly sensitive to adriamycin, while 14-3-3σ methylation-negative cell lines (either with wild-type or mutated p53) were resistant.
While no major overall impact can be attained with traditional chemotherapy in NSCLC, as explained before, it is clear that chemosensitivity and thus survival is individually predetermined. Nevertheless, in spite of the growing list of genetic abnormalities identified as being involved in DNA repair pathways and altered chemosensitivity in NSCLC patients, translational assays have not yet been developed for use in individualized chemotherapy.
It is an object of the present invention to provide predictors of response to chemotherapy, which can be a valuable clinical tool for use in the selection of optimal treatment modes, in particular for patients like those suffering from NSCLC, having such a poor survival rate and unpredictable chemosensitivity.